The long-term objectives of this proposal are to elucidate the mechanisms of activation and desensitization of the human beta2- adrenergic receptor (betaAR) in response to stimulation by the natural ligand epinephrine (adrenalin) and drug analogues of epinephrine. Epinephrine stimulation of the betaAR is involved in the control of many cellular processes such as relaxation of lung smooth muscle, the speed and force of contraction of heart muscle, and the control of glycogen metabolism and gluconeogenesis. Because of its many important roles, the betaAR is the target of many drugs such as albuterol and salmeterol that are mainstays in the treatment of asthma,. The action of epinephrine is rapidly attenuated or desensitized by a complex series of events that serve to shut the receptor down. These processes include phosphorylation by cAMP-dependent protein kinase and betaAR-specific kinases, the binding of a protein called beta-arrestin, and the movement of the betaAR from the plasma membrane into the cell's interior (endocytosis). The first aim of this proposal is to identify the amino acids in the betaAR that are phosphorylated both in the unstimulated, basal state and after simulation by strong agonists such as epinephrine and weak agonists such as albuterol using matrix-assisted laser desorption ionization time- of-flight (MALDI-TOF) mass spectrometry. Particular emphasis will be placed on determining the time and concentration dependency of the phosphorylations by the various agonists. All studies will be perfomed in cultured human embryonic kidney cells that are transfected with either the wild type betaAR or specially engineered epitope-modified betaARs. The second aim is to determine the functional effects of modifying betaAR domains proposed to be involved in desensitization by site- directed mutagenesis. Particular focus will be placed on those amino acids that are phosphorylated by PKA and betaAR-specific protein kinases, and that affect beta-arrestin binding, although we will also examine other domains possibly involved in growth factor regulation of the betaAR, in palmitoylation of the betaAR, and in binding to PDZ domains. The third aim is to examine the interrelationships of desensitization, phosphorylation, internalization and recycling of the betaAR by mathematical modeling using data accumulated from aims I and II as well as from additional studies of phosphorylation/dephosphorylation kinetics.